Test strips for determining coagulation factor activities

ABSTRACT

Test strips for determining the activity of a coagulation factor in a blood sample are provided. The strip comprises a support, a sample inlet port for deposition of a blood sample, and a reaction area comprising a blood coagulation reagent. The sample inlet port is connected to the reaction area, and the coagulation reagent comprises blood plasma deficient in the coagulation factor for which activity is to be measured, an ionic citrate source an ionic calcium source, and either one or more coagulation contact phase activator reagents and phospholipids or a mixture of tissue factor and phospholipids. The disclosure further relates to in vitro methods for measuring an activity of a coagulation factor.

The present invention relates to the field of blood parameters analysisand in particular to devices and tools for determining coagulationfactor activities. Thus, it relates to the field of medicine, diagnosis,as well as to tools and machines adapted to take measures in bloodsamples.

The following definitions are included herein merely for the purposes ofenhancing the understanding of the invention described herein.

The term “blood coagulation reagent” as defined herein, relates to acompound or group of compounds, as well as to mixtures of compositionsthat allow coagulation of a blood sample alone or in combination withother reagents, said other reagents commonly being ionic calcium source(i.e., organic and inorganic calcium salts), as well as plasmasartificially depleted in any of the coagulation factors; coagulationcontact phase activators, mixtures of tissue factor and phospholipids,and control samples including either blood or plasma comprising knownamounts of any of the coagulation factors and covering factor activitiesconsidered both normal and pathologic values, according to commonlyaccepted reference ranges in haematology.

The term “coagulation contact phase activator” or “surface contactactivators” (herein used interchangeably) relates to any compound thatpromotes activation of the intrinsic coagulation pathway by surfacecontact. This intrinsic pathway is also referred to as contactactivation pathway. The contact activation pathway begins with formationof the primary complex on collagen by high-molecular-weight kininogen(HMWK), prekallikrein, and FXII (Hageman factor). Prekallikrein isconverted to kallikrein and FXII becomes FXIIa. FXIIa converts FXI intoFXIa. Factor XIa activates FIX, which with its co-factor FVIIIa formsthe tenase complex, which activates FX to FXa. The minor role that thecontact activation pathway has in initiating clot formation can beillustrated by the fact that patients with severe deficiencies of FXII,HMWK, and prekallikrein do not have a bleeding disorder. Instead, thecontact activation system seems to be more involved in inflammations.Examples of contact phase activators include, thus, any of the naturalcompounds or complexes formed with collagen, HMWK, and prekallikrein.

Other examples of contact phase activators include artificial compoundsof polyanionic nature, such as kaolin of general formula Al₂Si₂O₅(OH)₄,and other silicates, or organic acids, such as ellagic acid. Thesecompounds are indeed mimics of cell surfaces or of tissue surfaces.

A “factor depleted plasma” refers to plasma usually from an artificialorigin from which one or more target proteins have been removed, forexample, by means of selective affinity immune-adsorption technologiesor e.g. chemically. Generally, they are human citrated plasmas and theymay be deficient plasmas in any of the following factors: II, V, VII, X,VIII, IX, XI, XII.

The expression “low levels of the coagulation factor to be measured” isto be understood as that the composition (i.e. plasma) has an amount ofa specified coagulation factor lower than the amount considered asnormal according to commonly accepted laboratory reference ranges. Theamount of a particular coagulation factor in plasma can be determinedboth by non-functional (antigen) and functional (factor activity) means;both measures must not necessarily coincide and can be expressed as apercentage referred to by a primary international standard establishedby the World Health Organisation (WHO). Focusing on factor activities,the factor present in a particular sample can be measured by comparingthe value of some of the specific physical property that will vary asthe clot is forming (which in turn will depend on the particular kind ofassay used in the laboratory to determine the amount of factor, i.e.chromogenic, optical, electrical, etc) with those of a calibration graphobtained from a calibrator with a known factor activity percentagereferred to the WHO primary standard properly diluted to yield severalcalibration points and covering a factor activity percentage range broadenough to determine either normal and pathologic values, so that aparticular variation in the physical property used to draft thecalibration curve can be converted in a percentage of factor activity.Obviously, sample measures must be based on the same physical property,reagents, equipment and general procedure followed in the case of thecalibration curve. Normal amounts of coagulation factors are thosedefined by a range of values including the values usually found innormal subjects. It is widely accepted that normal amounts (activities)may vary depending on the specific assay, reagents, equipment andprocedure used to determine factor activities, among races andpopulations within said races, among the people with different bloodgroups, and so do the activity values of the coagulation factors. Thisis why the value of any of the coagulation factor activity varyingtypically from 70% to 150% could be considered normal. In the presentdescription the expressions “amount/level of a coagulation factors” and“activity of a coagulation factor” are considered synonymous, becauseindependently of the determination (non-functional vs. functional) onecorrelates with the other On the other hand, it is also widely acceptedthat a value of any of the coagulation factor activity varying from 40%to 70% is to be considered as non-normal but also non-pathologic; andthat a value of any of the coagulation factor activity varying from 0%to 40% is considered non-normal and pathologic. A “pathologicalcoagulation factor deficiency” refers to a deficiency degree of aparticular factor implying a disease and/or with severe consequences forthe maintenance of life.

Ranges given, such as activities of coagulation factors, temperatures,times, sizes, and the like, should be considered approximate, unlessspecifically stated. The expression “between XX and YY” is consideredequivalent to the expression “from XX to YY”.

BACKGROUND ART

In the field of blood coagulation, it is known to determine theparameters known as Activated Partial Thromboplastin Time (APTT) and/orthe Prothrombin Time (PT). Both parameters give a value indicating thetime required for blood coagulation. In principle, these measures do notrequire any specialized personnel or special equipment and they can bedetermined in hospitals or non-specialized laboratories. Moreover,particular devices exist, even Point of Care (POC) devices fordetermining these parameters in the hospitals or at home.

Examples of these devices include the CoaguCheck Plus® (CCP®)commercially available from e.g. Boehringer Mannheim. It is a batterypowered, portable laser photometer with APTT reagents (for intrinsicpathway coagulation) and PT reagents (for extrinsic pathwaycoagulation). The reagents are disposed in a wafer-like disposableplastic reagent cartridge/strip. A sample of whole blood is applied toan application well in the cartridge and blood flows through capillaryaction to the reagent chamber.

Coagulation starts when the blood of the sample comes into contact withthe reagents and is considered to have stopped when the photometerdetects cessation of blood flow. The detection is based on sensing thevariation in light scatter from red cells. The time between bloodapplication and blood clotting is measured and converted to the plasmaequivalents APTT or PT.

The document WO9013034 discloses a test strip comprising an orifice orarea adapted to receive a sample of whole blood, a capillary trackleading the sample to a zone comprising the reagents for initiatingcoagulation and for the in situ measure of APTT. The strip is alsoprovided with a venting port. The aim of the device is the determinationof blood flow stoppage independently of the blood haematocrit, which hasbeen reported as an inconvenient in test strips using blood samples asfluid test.

Determination of APTT or PT, as well as of other blood parameters, is ofgreat relevance for patients receiving anti-coagulant treatments. In thesame way, coagulation time is a critical aspect in intensive care unitsfor the monitoring of heparin treatment and in preoperative assessmentof blood coagulation. As a general rule, APTT or PT may be easilydetermined in normal conditions, which means that the subject (patient)does not have any inherited or acquired coagulation factor deficiency.APTT and/or PT may be altered when the subject has a coagulation factordeficiency, making further diagnostic tests necessary. In thesescenarios patients are redirected to more specialized hospitals oranalytical laboratories, which elongate the diagnosis time. This alsosupposes an uncomfortable procedure for the patient. Anotherdisadvantage is that relatively high amount of blood (namely the furtherprocessed plasma) may be required.

The standardised mode to measure the deficiency of one or morecoagulation factors can be summarized as follows. A plasma test sampleis firstly diluted with a buffer, and then mixed with factordepleted-plasma which is deficient only in the coagulation factor thatis going to be detected in the test sample, and comprising all othercoagulation factors. Next, reagents either from the extrinsic orintrinsic coagulation pathway needed to start coagulation are added tothe mixture. Coagulation is then only dependent on the limitingcoagulation factor, corresponding to the one being measured in the testsample. A modification of this standardised method is in particulardisclosed for factor V activity in the document WO9720066. The documentWO9720066 discloses the way for determining if a patient is susceptibleof suffering a thromboembolism. Analysis is performed using plasma anddetermining the ratio between factor V activity level without activatedprotein C (APC) and the factor V activity level with APC. Factor Vdepleted plasma is added to increase the specificity of the test.

Among the commercial tests adapted for detecting coagulation factordeficiencies, the test known as Actin FS® from Baxter Diagnostics Inc.is one of the multiple examples commonly used in the specializedlaboratories to test coagulation factor activities in plasma samples.The test includes all the reagents for an APTT determination (purifiedsoy phosphatides, ellagic acid activator and calcium) in liquid form andready for use. It allows analysing factor defects of factors VIII, IX,XI, and XII with high sensitivity and specificity.

As above exposed all these test are performed on plasma and requirevenous blood samples.

It is an object of the present invention to provide methods and devicesthat at least partially solve one or more of the aforementionedproblems.

SUMMARY

In a first aspect, a test strip for determining the activity of acoagulation factor in a blood sample is provided. The strip comprises asupport, a sample inlet port for deposition of a blood sample, and areaction area comprising a blood coagulation reagent. The sample inletport is connected to the reaction area, and the coagulation reagentcomprises plasma deficient in the coagulation factor for which activityis to be measured, an ionic calcium source, an ionic citrate source, andeither one or more coagulation contact phase activator reagents andphospholipids or a mixture of tissue factor and phospholipids.

In the present invention “plasma” refers to blood plasma, which is thestraw-colored/pale-yellow liquid component of blood that normally holdsthe blood cells in whole blood in suspension. It may be mostly water(92% by volume), and may contain dissolved proteins (i.e.—albumins,globulins, and fibrinogen), glucose, clotting factors, electrolytes(Na+, Ca2+, Mg2+, HCO3-Cl— etc.), hormones and carbon dioxide (plasmabeing the main medium for excretory product transportation). Plasma isprepared by spinning a tube of fresh blood containing an anticoagulantin a centrifuge until the blood cells fall to the bottom of the tube.

In accordance with this aspect, a sample of whole blood (obtained bye.g. capillary puncture of a fingertip of a patient) may be introducedin the sample inlet port. The sample will reach the reaction areacontaining plasma deficient in the coagulation factor whose activity isgoing to be measured. By providing this plasma deficient in a specificfactor (and importantly, normal amounts of the others), the amount offactor present in the patient's blood sample will become the reactionlimiting element and thus the coagulation time will only be dependent ofthis variable. By measuring the coagulation time, the activity of theselected coagulation factor may be determined.

Thus, the proposed strips provide a way to correlate a clotting timewith the corresponding percentage of activity of the clotting factorthat is actually being measured.

A test strip is herein provided which may be combined with knownequipment for determining coagulation time, such as the CoaguCheck Plus®mentioned before (or slightly modified versions thereof) to measureclotting factor activities. The equipment may be able to determine achange in phase based e.g. on IR emissivity/reflectivity or otherwise.

In case of coagulation factors linked to the extrinsic pathway of bloodclotting, the coagulation reagent may comprise a mixture of tissuefactor, calcium and phospholipids. In case of coagulation factors linkedto the intrinsic pathway of blood clotting, the coagulation reagent maycomprise coagulation contact phase activator, calcium and phospholipids.The coagulation reagent may be provided in e.g. dried or lyophilizedform.

Inventors propose a test strip that allows measuring any coagulationfactor deficiency using low amounts of whole blood, even of capillaryblood.

The strips may thus easily be used as POC devices in hospitals or evenby a patient in his or her house. The strips may even function well withlow sample volumes. They may be portable and small sized. In addition,the strips may be manufactured using known and relatively simpleprocedures, thus implying low production costs.

Thus, they represent the provision of a long-felt need and they mayimprove not only a patient's quality of life, but may also improvediagnostic of diseases related to the impairment of coagulationpathways, such as haemophilia, by hospital personnel. Hence and of greatinterest, any therapeutic decision of administering or not a particulardrug or coagulation factor may be reached in the shortest possible time.

In some embodiments, in the reaction area one or more of the ingredientsis separated from another ingredient. The separation between ingredientsmay include a physical barrier (e.g. a wall of a compartment) or the(dried or lyophilized) ingredients may be immobilized in the reactionarea at a distance from each other. One or more capillary tracks may beprovided between compartments or ingredients to ensure proper mixture ofingredients, and in a predetermined order.

In some examples, the reaction area may comprise a first portion, asecond portion, and third portion, the portions being substantiallyseparated from each other, and the first portion comprises the plasmadeficient in the coagulation factor for which activity is to be measuredand an ionic citrate source, the second portion comprises one or morecontact phase activator reagents and phospholipids, and the thirdportion comprises an ionic calcium source, A separation of theingredients ensures that mixture may take place in a predeterminedorder. The order herein described is particularly suitable for factorsinvolved in the intrinsic pathway. In another example, even the ioniccitrate source may be in a separate portion, thus an additional portionmay comprise the ionic citrate source separated from the plasmadeficient in the coagulation factor for which activity is to bemeasured.

In some other examples, the reaction area may comprise a first portionand a second portion separated from each other, and the first portioncomprises the plasma deficient in the coagulation factor for whichactivity is to be measured and an ionic citrate source, and the secondportion comprises a mixture of an ionic calcium source, tissue factorand phospholipids. Such a test strip may be particularly suitable forfactors involved in the extrinsic pathway. As above, in another examplethe ionic citrate source may be in a separate portion, thus anadditional portion may comprise the citrate source separated from theplasma deficient in the coagulation for which activity is to bemeasured.

In some embodiments, the sample inlet port may be connected with thereaction area by a capillary track. Capillary tracks are known in thefield of test strips. The separation of the sample inlet port from thereaction area ensures that clotting time may be reliably measured bydetermining a first phase change from solid to liquid (i.e. when bloodof the patient's blood sample reaches the reaction area) and then fromliquid to solid (i.e. when clotting has taken place).

In some embodiments, a test strip may further comprise a control fluidinlet port and a normal blood reaction area comprising blood or plasmacontaining standard (normal) known amounts of any of the coagulationfactors (i.e. amounts correlating with or giving rise to an activityfrom 70% to 150%), wherein the second reaction area is connected to thecontrol fluid inlet port, optionally through a capillary track.

Optionally, the test strip may furthermore or alternatively comprise acontrol fluid inlet port and a depleted blood reaction area comprisingblood or plasma containing known levels (amounts) of the coagulationfactor to be measured, said known levels of coagulation factor beinglower than a reference value and/or out of a reference range, saidreference value including standard (normal) amounts or ranges ofactivities (i.e. from 70% to 150%) and non-normal but at the same timenon-pathological activities (i.e. from 40% to 70%). By out of anyreference values or range is to be understood that according to saidreference ranges the value is a pathological one. In this specific case,it is a value lower than the lowest limit of the reference range.

In a particular embodiment, the depleted blood reaction area comprisesknown levels of coagulation factor correlating with an activity beinglower than 40%.

The provision of one or more further reaction areas comprising eitherblood/plasma with normal amounts of coagulation factor and blood/plasmawith a relatively low amount of the coagulation factor to be measuredmay aid in improving the reliability of the measurements. The blood inthese areas may be provided in dried or lyophilized form. The controlfluid may be e.g. a physiological serum or buffer, or even distilledwater in case the reaction areas comprise already buffered solutions.When the control fluid reaches the reaction area, a phase change fromsolid to liquid may be registered. When clotting occurs, a phase changefrom liquid to solid may then again be measured. Since the “amount” ofthe coagulation factor in these control areas is known, its coagulationtime is known as well. It may thus be checked whether the coagulationtime registered for the control areas coincides with the theoreticalclotting time. If it does, one thus is able to conclude that theequipment used for measuring clotting is functioning properly. In aparticular embodiment, reaction areas may be in the form of wells.

In some embodiments, the test strips may comprise a computer readablememory comprising data linking coagulation times that may be measured toactivities of the coagulation factor to be measured. The data may e.g.be in the form of a coagulation curve or a look-up table indicating theactivity level of a coagulation factor as a function of a coagulationtime. Suitable equipment may read the memory and be able to indicatedirectly the level of activity of the coagulation factor underinvestigation. Coagulometers adapted for reading computer readablememory integrated in a test strip may be used.

In another aspect, an in vitro method for measuring an activity of acoagulation factor of the intrinsic or extrinsic pathway is provided.The method comprises contacting an isolated capillary blood sample froma test subject with a composition comprising a coagulation contact phaseactivator, a coagulation factor depleted plasma, an ionic citratesource, and an ionic calcium source, or alternatively, contacting thesample with a mixture of tissue factor and phospholipids, a coagulationfactor depleted plasma, an ionic citrate source, and an ionic calciumsource; determining the time required for coagulation of said sample;and correlating said coagulation time with a particular activity bycomparison with a coagulation function, said the coagulation functionrepresenting the relation between coagulation time and the activity of acoagulation factor.

In some embodiments, the method may be used for rendering a diagnosiswherein, if the time required for coagulation is higher than a referencevalue or range from subjects with no coagulation factor deficiency, itis indicative of pathological coagulation factor deficiency. Thereference value or reference range is in particular the time or range oftimes correlating with non-pathologic activities. Such a reference mayinclude normal values and also non-normal values but considerednon-pathological for a particular coagulation factor. Thus, if the timerequired for coagulation is higher than a reference value or range, thismeans that the activity of the factor is lower than the reference valueand, it is indicative of pathological coagulation factor deficiency.

In a particular embodiment, the activity of the coagulation factor islower than 40% in case of a deficiency of any of the tested coagulationfactors.

The method may also be used for diagnosing non-normal and at the sametime non-pathological activities of a particular coagulation factor.This is the particular case when the activity detected is in the rangeof values from 40% to 70%. Although it depends on the tested factor, formost of them this range of activities is indicative of coagulationfactor deficiency (not normal amounts; 70%-150%) but of non-pathologicalkind. Determination of non-normal but non-pathological activities of afactor allows deciding on a particular medical treatment including thefollow-up of the patient without treatment.

In a particular example of the in vitro diagnosis method of theinvention, the coagulation factor deficiency is selected from the groupconsisting of deficiencies of coagulation Factor V (FV), deficiencies ofcoagulation Factor VII (FVII), deficiencies of coagulation Factor VIII(FVIII), the deficit or deficiency of which causes hemophilia A,deficiencies of coagulation Factor IX (FIX) the deficit or deficiency ofwhich causes hemophilia B, deficiencies of coagulation Factor X (FX),deficiencies of coagulation Factor XI (FXI), and deficiencies ofcoagulation Factor XII (FXII).

These deficiencies may be congenital or me be acquired coagulopathies(deficiencies) of different origins. Illustrative examples includecoagulation factor synthesis deficiencies or inhibition such as inanticoagulant therapy (heparin, low molecular weight heparins, warfarin,coumarin derivatives, dicoumarins, etc.), or in severe hepatic failureor presence of acquired inhibitors. Acquired coagulopathies leading tocoagulation factor deficiencies may be due to an exaggerated consumptionof coagulation factors, thus making them not available to form the clotin a bleeding lesion. This mechanism occurs for example in thedisseminated intravascular coagulation syndrome due to consumptionoccurring in multiple illnesses such as in severe sepsis, wherein theformation of multiple microthrombi diminishes the levels of coagulationfactors. Another example is in blood invasion by tissue factor such asplacental release; in the retention of a dead fetus; in multiple traumaswith the crushing of tissues; in poisonous snake bites, etc. In allthese diseases or conditions, the consumption of coagulation factors isworsened by lysis of the fibrin of numerous microthrombi due to theaction of plasmin, which are antiplatelets and anticoagulants.

In a particular example of the method, step (i) is carried out bycontacting an isolated capillary blood sample from a test subject with acomposition comprising a coagulation contact phase activator, acoagulation factor depleted plasma, an ionic citrate source, and anionic calcium source.

This embodiment allows the determination of the activity of acoagulation factor of the intrinsic pathway. In another particularexample of the method for measuring the activity of a coagulation of theintrinsic pathway, step (i) may be carried out by:

(a) first contacting an isolated capillary blood sample from a testsubject with a composition comprising a coagulation factor depletedplasma and an ionic citrate source to obtain a primary mixture;(b) further contacting the primary mixture of step (a) with acoagulation contact phase activator, an ionic calcium source andphospholipids; and(c) contacting the mixture of step (b) with an ionic calcium source.

In some embodiments, step (b) may be performed at a temperature fromapproximately 35° C. to 39° C. and for a time from 15 seconds to 5minutes. It is in step (b) that the mixture contacts the reagents knownas APTT reagents. In some other embodiments, optionally in combinationwith any embodiment described further below or above, step (c) may becarried out at a temperature from 35° C. to 39° C. Particular examplesof coagulation factors of the intrinsic pathway which activity may bedetermined by this method include factor VIII, IX, XI and XII.

All these steps (a) to (c) may be performed in a test strip providedwith a reaction area with multiple compartments. In each compartmentthere may be provided the compounds or compositions in lyophilized orliquid form. Mixture of the sample with the compounds or compositionsmay be achieved capillarity.

In a particular example of the method, step (i) may be carried out bycontacting an isolated capillary blood sample from a test subject with acomposition comprising a mixture of tissue factor and phospholipids, aplasma deficient in the coagulation factor for which activity is to bemeasured, an ionic citrate source, and an ionic calcium source.

This embodiment allows the determination of the activity of acoagulation factor of the extrinsic pathway. In another particularexample of the method for measuring the activity of a coagulation of theextrinsic pathway, step (i) may be carried out by:

(a) first contacting an isolated capillary blood sample from a testsubject with a composition comprising a coagulation factor depletedplasma and an ionic citrate source to obtain a primary mixture; and(b) further contacting the primary mixture of step (a) with a mixture oftissue factor and phospholipids, an ionic calcium source andphospholipids.

In an embodiment, step (b) may be performed at a temperature from 35° C.to 39° C. This step (b) is, the step in which the mixture of step (a)enters into contact with the known PT reagents. Particular examples ofcoagulation factors of the extrinsic pathway which activity may bedetermined by this method include factor II, V, VII and X.

All these steps (a) to (b) may be performed in a test strip providedwith a reaction area with multiple compartments. In each compartment thecompounds or compositions may be provided in lyophilized or liquid form.Mixture of the sample with the compounds or compositions may beachieved, by capillarity.

In any of the examples of the method, the step (ii) of determining thetime required for blood coagulation, may start when in any of theoptions the sample (or mixtures) enter into contact with the calciumsource. Afterwards, the method may include step (iii) of correlatingblood coagulation time with a particular activity.

Any of the methods hereinbefore described allows detecting if a subjectis suffering from any coagulation factor deficiency using low volumesamples (capillary blood) in contrast with the prior art tests.

Additional objects, advantages and features of the invention will becomeapparent to those skilled in the art upon examination of the descriptionor may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Particular embodiments of the present invention will be described in thefollowing by way of non-limiting examples, with reference to theappended drawings, in which:

FIGS. 1 a and 1 b schematically illustrate top views of differentexamples of test strips;

FIG. 1 c schematically illustrates a coagulation curve which may be usedin combination with the example of FIG. 1 b;

FIG. 2 a schematically illustrates a top view of another example of atest strip;

FIG. 2 b schematically illustrates a coagulation curve which may be usedin combination with the example of FIG. 2 a; and

FIG. 3 is a schematic perspective view of the strip according to yetanother example.

DETAILED DESCRIPTION

FIG. 1 a schematically illustrates a top view of a test strip accordingto a first example. A strip 10 may comprise a support in which a sampleinlet port 12 is provided. Also a reaction area 14 is indicated. A track13 may connect the sample inlet port with the reaction area 14.

The track 13 may be a capillary track. In alternative examples, thetransport of the blood sample from the deposition area 12 to thereaction area 14 may be based on a different principle, such as e.g.pumping or suction.

The support may comprise one or more layers of semi-rigid plastic. A toplayer may be provided that closes off the reaction area and/or thedeposition area from the top. A patient, doctor or laboratory personnelstaff may deposit a drop or few drops of whole blood in the depositionarea 12 (e.g. after uncovering). In one possible implementation, theblood may have been extracted through a capillary puncture in thefingertip.

The layers may be made suitable for the measurement method employed. Forexample, they may be substantially transparent or translucent if themethod for determining clotting is based on e.g. lightabsorption/reflection. If the measurement method is based on e.g. anelectrical characteristic (such as e.g. the impedance), suitablematerials may be chosen and a suitable electric circuit may be providedin the support.

The reaction area may comprise blood coagulation reagent which may varydepending on the clotting factor to be investigated. The clotting factor(or “blood coagulation factor”) for which the activity is measured maybe selected from the group consisting of factor II, V, VII, X, VIII, IX,XI and XII.

Depending on the factor to be investigated, the blood coagulationreagent may comprise plasma deficient in the selected coagulationfactor, an ionic calcium source and either one or more coagulationcontact phase activator reagents and phospholipids (for factors relatedto the intrinsic pathway) or a mixture of tissue factor andphospholipids (for factors related to the extrinsic pathway).

The coagulation contact phase activators may be selected from the groupconsisting of polyanionic compounds, organic acids and mixtures thereof.The calcium source may be a calcium salt selected from the groupconsisting of calcium chloride, calcium acetate, calcium carbonate,calcium glubionate, calcium gluconate, calcium hydroxide, calciumnitrate, calcium sulfonate, calcium phosphate, and mixtures of thesesalts. The ionic citrate source may be sodium citrate and/oracid-citrate-dextrose. In a preferred embodiment the ionic citratesource is sodium citrate.

The coagulation reagent may be in liquid, semi-solid or solid form. Thecoagulation reagent may be lyophilized. Alternatively, the coagulationreagent may be otherwise immobilized in the reaction area. Once thewhole blood sample of the patient reaches the reaction area, a localchange may be measured, in particular in the case of a lyophilizedreagent, a phase change from solid to liquid may be measured. When aphase change from liquid to solid is registered coagulation has takenplace. The coagulation time may thus be derived from the time passedbetween the first change and the second change. Other known methods fordetermining coagulation times may also be used.

Any suitable equipment such as a coagulometer for this sort ofdetermination may be used and may be based e.g. on optical principles(reflexivity/emissivity), mechanics, inductance, electric resistance orimpedance and other (or combinations thereof). In some examples,portable coagulometers may be used. The test strip may be introducedinto the coagulometer either before or after deposition of the wholeblood sample. The dimensions of the test strip may be determined inaccordance with the equipment used for analysis.

FIG. 1 a further illustrates an example of how ingredients of thecoagulation reagents may be separated in a reaction area. In theschematically illustrated example, the reaction area may comprise afirst portion 14 a, a second portion 14 b, and a third portion 14 c.

The portions 14 a, 14 b and 14 c may be substantially separated fromeach other: this may be achieved e.g. by creating separate compartments.Separate compartments may be particularly useful when the ingredientsare provided in liquid form. In another example, the ingredients may beprovided in dried or lyophilized form. If properly immobilized, separatecompartments may not be necessary.

In this particular example, the first portion may comprise the plasmadeficient in the coagulation factor for which activity is to be measuredand an ionic citrate source. The second portion may comprise one or morecontact phase activator reagents and phospholipids, and the thirdportion may comprise an ionic calcium source,

A separation of the ingredients ensures that mixture may take place in apredetermined order. Capillary tracks 15 a and 15 b connect the separateportions. The length of the capillary track may determine the time ittakes for the mixture to reach a next portion. In this sense, properdimensioning of the capillary track can ensure that mixture ofingredients take place before the next ingredient is reached. In otherexamples, other mechanisms such as suction or pumping may be used toconnect the separate ingredients. The order described in this example isparticularly suitable for factors involved in the intrinsic pathway.

In an alternative-non-illustrated example, two separate portions couldbe provided for the extrinsic pathway factors. The first portion in suchan example may comprise the plasma deficient in the coagulation factorfor which activity is to be measured and an ionic citrate source, andthe second portion may comprise an ionic calcium source and a mixture oftissue factor and phospholipids.

FIG. 1 b illustrates another example of a test strip 10. The test stripcomprises a sample deposition area 12, track 13 and reaction area 14similar to the one showed in the example of FIG. 1 a. Additionally, thestrip in this example may comprise a control fluid deposition area 16,and a track 17 connecting the deposition area 16 with a control reactionarea 18.

The control reaction area 18 may comprise a lyophilized blood or plasmasample of known composition. In an example, the blood or plasma samplemay comprise normal amounts of the coagulation factors (i.e. withactivities from 70% to 150%) or alternatively the blood or plasma samplemay have a relatively low known amount of the coagulation factor to beinvestigated, being said relatively low known amount an amount (or levelof coagulation factor) lower than and out of any normal amount or rangeof normal amounts (i.e. with activities of 70%-150%), or lower than andout of a non-normal and non-pathological amount or range (i.e. withactivities from 40% to 70%). Thus, in another example, the blood orplasma sample may have an amount of coagulation factor activity lowerthan 40%. This means that the factor activity of the blood controlsample is lower than 40%, which reflects a pathological value.

In some examples, the control reaction area(s) may comprise a mixture ofblood or plasma with a known amount of coagulation factor, a citratesource, one or more contact phase activator reagents and phospholipids,and an ionic calcium source. Such an example may be suitable for factorsinvolved in the intrinsic pathway. In some other examples more suitablefor the extrinsic pathway factors, the control reaction area(s) maycomprise a mixture of blood or plasma with a known amount of coagulationfactor, a citrate source, an ionic calcium source and a mixture oftissue factor and phospholipids.

In either case, the control reaction area may serve to check or confirmthe results obtained in the reaction area. Reference may be had to FIG.1 c schematically illustrating a coagulation curve 30 showing therelation between coagulation time and a level of activity of a specificcoagulation factor.

In some examples, a similar separation of ingredients as illustrated inFIG. 1 a for the reaction area may be used in such a control reactionarea. A first portion could comprise the blood or plasma with normalamounts of coagulation factor and the citrate source; or blood or plasmawith a relatively low amount of the coagulation factor and the citratesource. If the coagulation factor to be measured is from the intrinsicpathway, the second portion may comprise one or more contact phaseactivator reagents and phospholipids, and the third portion may comprisean ionic calcium source. If the coagulation factor to be measured isfrom the extrinsic pathway, with two portions may be enough and saidsecond portion may comprise an ionic calcium source and a mixture oftissue factor and phospholipids. As explained above for the reactionarea, separate compartments may be particularly useful when theingredients of control areas are provided in liquid form. In anotherexample, the ingredients may be provided in dried or lyophilized form.If properly immobilized, separate compartments may not be necessarywhile still maintaining separation between different ingredients.

With this kind or pattern of ingredient distribution in the controlreaction areas the method of the invention may be accomplished in thesame experimental conditions as in the reaction area, wherein the sampleof the patient is going to be tested. That is, in the same order, andsame incubation times with the particular ingredients. If theingredients in the control reaction areas are in dried or lyophilizedform, they are rehydrated or allowed to evolve to a liquid form with thecontrol fluid (physiological serum or buffer, or even distilled water incase the reaction areas comprise already buffered solutions).

In an example, the control fluid may be a physiological serum, or aphysiological buffer of some kind. Through e.g. capillary action, thecontrol fluid may reach the control reaction area 18 and may bring abouta phase change from solid to liquid. After clotting, a phase change fromliquid to solid may occur. Since the amount of the coagulation factor inthe control sample is known, its theoretical coagulation time whichshould lie on coagulation curve 30 is known as well. If in a test it isfound that the control sample factor activity indeed lies within somepreviously established limits considered acceptable then this is anindication that the test equipment is working properly. If on the otherhand, a deviation from those limits is found, this may indicate amalfunction of some sort.

Alternatively to a coagulation curve such as the one illustrated in FIG.1 c, the relation between clotting time and level of activity of thecoagulation factor may be stored in alternative manners, e.g. in theform of a look-up table or in the form of a mathematical equation.

FIG. 2 a schematically illustrates a further example of a test strip 10.In this example, three capillary tracks may be arranged. A sample inletport 12 may be connected to a reaction area 14 via a capillary track 13.A first control fluid inlet port 16 may be connected to a first controlreaction area 18 via a capillary track 17. A second control fluid inletport 21 may be connected to a second control reaction area 23 via acapillary track 22.

The first control reaction area 18 may be a standard blood reaction areacomprising blood containing a known normal amount of the coagulationfactor whose activity is to be measured, and also including normalamounts of any of the other coagulation factors.

The second control reaction area 23 may be a depleted blood reactionarea comprising blood with a relatively low (but known) amount of thecoagulation factor to be measured and substantially normal amounts ofthe other coagulation factors. The relatively low known amount is anamount (or level of coagulation factor) lower than and out of any normalamount or range of standard amounts. Thus, in another example, thedepleted blood reaction area may comprise blood with an amount ofcoagulation factor with an activity lower than 70%. This means that theactivity of the blood control sample is lower than 70%.

Schematically illustrated is a chip 25 comprising a computer readablememory. The memory may be read by suitable equipment, e.g. the sameequipment used for determining the clotting time.

A control fluid may be deposited in the control fluid inlet ports orareas 16 and 21. Substantially at the same time a blood sample may bedeposited in the sample inlet port 12. The reaction strip may then beinserted into suitable laboratory equipment capable of determiningclotting and capable of reading chip 25. Alternatively and depending onthe equipment used, the strip may have been inserted into the equipmentprior to deposition of the sample and control fluid.

Once the blood sample reaches the reaction area, clotting may begin.Once the control fluid reaches the control reaction areas, thelyophilized samples may change from a solid state to a liquid state andthen clotting may begin in the reaction areas. Other known methods fordetermining coagulation times may also be used.

In accordance with FIG. 2 b, in a single strip a reliable indication ofthe level of activity of the coagulation factor may be found. Theclotting times found in the two control reaction areas are expected tolie between previously established acceptability limits. This curve andinformation on the composition of the samples in areas 18 and 23 may bestored in the memory of chip 25. If the clotting times found in thecontrol areas both lie within the limits, it may be confirmed that thelaboratory equipment and reagents used in functioning correctly and thatthe test strip is not defective.

The clotting time found in the reaction area 14 may thus reliablyindicate the level of activity of the coagulation factor beinginvestigated.

Alternatively to storing the coagulation curve (or similar data) on achip incorporated in the test strip, the information may be previouslystored in the laboratory equipment used. In yet a further alternative,for each manufacturing batch of test strips, a single test strip withsuch a chip or computer readable memory may be provided. The readingsfrom this chip may be held to be representative for the complete batch,as it may be assumed that a manufacturing batch will generally comprisethe same or very similar lyophilized blood samples and/or reagents ineach strip.

FIG. 3 illustrates a further alternative example of a test strip. Theprinciples upon which this example is based are largely the same asthose illustrated and explained with reference to FIGS. 2 a and 2 b.However, a single control fluid inlet port 16 is connected by abifurcated track or channel 17 which leads to two control reaction areasthrough partial channels 17 a and 17 b. As in the previously illustratedexample, the first control reaction area 18 may comprise bloodcontaining a normal amount of the coagulation factor for which activityis to be measured and the second control reaction area 23 may be adepleted blood reaction area comprising blood with a relatively lowamount of the coagulation factor to be measured.

Throughout the description and claims the word “comprise” and variationsof the word, are not intended to exclude other technical features,additives, components, or steps. Furthermore, the word “comprise”encompasses the case of “consisting of”.

Although only a number of particular embodiments and examples of theinvention have been disclosed herein, it will be understood by thoseskilled in the art that other alternative embodiments and/or uses of theinvention and obvious modifications and equivalents thereof arepossible. Furthermore, the present invention covers all possiblecombinations of the particular embodiments described. Thus, the scope ofthe present invention should not be limited by particular embodiments,but should be determined only by a fair reading of the claims thatfollow.

1. A test strip for determining the activity of a coagulation factor ofthe intrinsic or extrinsic pathway in a blood sample, said stripcomprising: a support disposed on which are: a sample inlet port fordeposition of a blood sample, and, a reaction area comprising a bloodcoagulation reagent, wherein the sample inlet port is connected to thereaction area, and wherein the coagulation reagent comprises thefollowing ingredients: plasma deficient in the coagulation factor forwhich activity is to be measured, an ionic citrate source, an ioniccalcium source, and either one or more coagulation contact phaseactivator reagents and phospholipids or a mixture of tissue factor andphospholipids.
 2. The test strip according to claim 1, including one orboth: wherein in the reaction area one or more of the ingredients areseparated from another ingredient, or further comprising a capillarytrack between separate ingredients.
 3. (canceled)
 4. The test stripaccording to claim 2, wherein the reaction area comprises a firstportion, a second portion, and a third portion, the first, second andthird portions being substantially separated from each other, andwherein the first portion comprises the plasma deficient in thecoagulation factor for which activity is to be measured and an ioniccitrate source, the second portion comprises one or more contact phaseactivator reagents and phospholipids, and the third portion comprises anionic calcium source.
 5. The test strip according to claim 2, whereinthe reaction area comprises a first portion and a second portionseparated from each other, and wherein the first portion comprises theplasma deficient in the coagulation factor for which activity is to bemeasured and an ionic citrate source, and the second portion comprisesan ionic calcium source and a mixture of tissue factor andphospholipids.
 6. (canceled)
 7. The test strip according to claim 1,including one or more of: wherein the reaction area comprises separatecompartments; or wherein the sample inlet port is connected with thereaction area by a capillary track; and, wherein the coagulation reagentis one or both lyophilized or immobilized in the reaction area.
 8. Thetest strip according to claim 1, further comprising a control fluidinlet port and a normal blood reaction area comprising blood or plasmacontaining a normal, known amount of the coagulation factor for whichactivity is to be measured, said normal amounts giving rise to anactivity from 70% to 150%, wherein the normal blood reaction area isconnected to the control fluid inlet port, optionally through acapillary track.
 9. The test strip according to claim 1, furthercomprising a control fluid inlet port and a depleted blood reaction areacomprising blood or plasma containing a known pathological level of thecoagulation factor to be measured, said known level of coagulationfactor being lower than a reference value and/or out of a referencerange, wherein said reference value or range is defined by activitiesfrom 40% to 150%.
 10. The test strip according to claim 8, comprising asingle fluid inlet port connected through capillary tracks to the normalblood reaction area and a depleted blood reaction area.
 11. The teststrip according to claim 1, further comprising a computer readablememory comprising data linking coagulation time to activity of thecoagulation factor to be measured.
 12. The test strip according to claim1, wherein the coagulation factor for which activity is measured is inturn selected from the group consisting of factor II, V, VII, X, VIII,IX, XI and XII.
 13. (canceled)
 14. The strip according to claim 1,wherein the coagulation contact phase activator is selected from thegroup consisting of polyanionic compounds, organic acids, and mixturesthereof.
 15. The strip according to claim 14, wherein the polyanionicactivator compounds are silicates.
 16. The strip according to claim 15,wherein the coagulation contact phase activator is kaolin.
 17. The stripaccording to claim 14, wherein the coagulation contact phase activatoris ellagic acid.
 18. The strip according to claim 1, characterized inthat the calcium source is a calcium salt selected from the groupconsisting of calcium chloride, calcium acetate, calcium carbonate,calcium glubionate, calcium gluconate, calcium hydroxide, calciumnitrate, calcium sulfonate, calcium phosphate, and mixtures of thesesalts.
 19. An in vitro method for measuring an activity of a coagulationfactor of the intrinsic or extrinsic pathway comprising (i) contactingan isolated capillary blood sample from a test subject with acomposition comprising a coagulation contact phase activator, acoagulation factor depleted plasma, an ionic citrate source, and anionic calcium source, or alternatively, contacting the sample with amixture of tissue factor and phospholipids, a plasma deficient in thecoagulation factor for which activity is to be measured, an ioniccitrate source, and an ionic calcium source; (ii) determining the timerequired for blood coagulation of said sample; and (iii) correlatingsaid blood coagulation time with a particular activity by comparisonwith a coagulation function, said coagulation function representing therelation between coagulation time and the activity of a coagulationfactor.
 20. (canceled)
 21. The method according to claim 19, wherein oneor both: the coagulation function is defined as a mathematical equation,or a look-up-table, or a coagulation curve; and, if the time requiredfor blood coagulation is higher than a reference value from a subjectwith no pathological coagulation factor deficiency, it is indicative ofpathological coagulation factor deficiency.
 22. The method according toclaim 19, wherein the coagulation factor is selected from the groupconsisting of Factor II (FII), Factor V (FV), Factor VII (FVII), FactorX (FX), Factor VIII (FVIII), Factor IX (FIX), Factor XI (FXI), andFactor XII (FXII).
 23. The method according to claim 19, wherein one orboth A and B: A: wherein the coagulation factor is of the intrinsicpathway and step (i) is carried out by: (a) first contacting an isolatedcapillary blood sample from a test subject with a composition comprisinga coagulation factor depleted plasma and an ionic citrate source toobtain a primary mixture; (b) further contacting the primary mixture ofstep (a) with a coagulation contact phase activator, an ionic calciumsource and phospholipids; and (c) contacting the mixture of step (b)with an ionic calcium source; and B: wherein the coagulation factor isof the extrinsic pathway and step (i) is carried out by: (a) firstcontacting an isolated capillary blood sample from a test subject with acomposition comprising a coagulation factor depleted plasma and an ioniccitrate source to obtain the primary mixture; and (b) further contactingthe primary mixture of step (a) with a mixture of tissue factor andphospholipids, an ionic calcium source and phospholipids.
 24. (canceled)25. The test strip according to claim 9, comprising a single fluid inletport connected through capillary tracks to a normal blood reaction areaand the depleted blood reaction area.